Title

Author

Date of Award

5-2017

Document Type

Honors Thesis

Degree Name

Bachelor of Science

Department

Biological Science

Advisor/Committee Chair

Robert Osuna

Committee Member

Gabriel Fuchs

Abstract

DksA, a global gene regulator, binds to RNA po lymerase to regulate transcription initiation in Escherichia coli . It negatively regulates transcription of some genes, such as those for ribosomal RNAs and fis , and positively regulates transcription of other genes, including genes involved in amino acid biosynthesis. Little is known about how DksA itself is regulated. Current work in our lab has shown three major promoters, a σ 70 dependent P 1 promoter and two σ 38 (RpoS) - dependent promoters (P 2 and P 3 ), regulate the transcription of dksA . More is current ly known about the regulation of the P 1 than of the P 2 and P 3 promoters. This work focuses on increasing our understanding of how dksA P 3 promoter may be regulated. More specifically, we wished to investigate 1) if DksA regulates the transcription of dksA P 3 , 2) if dksA P 3 requires a - 35 promoter region, and 3) if there are upstream promoter sequences that are involved in the regulations of transcription of dksA P 3 . To this end, I generated a nested set of deletions of the region upstream of dksA P 3 using PCR, cloned the DNA fragments on a plasmid DNA to fuse the promoters to the lacZ reporter gene, moved the lacZ fusions onto a l bacteriophage DNA by homologous recombination, and then integrated the recombinant l bacteriophage DNA onto the E. coli chromoso me as a single copy. Results from b - galactosidase assays in wild type and ∆ dksA strains showed that: 1) DksA stimulates transcription of the dksA P 3 promoter, 2) transcription of dksA P 3 does not require a - 35 promoter sequence, and 3) the region upstream of - 100 and of - 75 (relative to the dksA P 3 transcription start site), appear to be involved in negative and positive regulation of dksA P 3 , respectively. Inspection of the DNA sequences in these regions revealed potential binding sites for the transcription regulators SoxS, LexA in the re gion 3 upstream of - 100, and for cpxR in the region upstream of - 75. This work has broadened our understanding of how dksA P 3 may be regulated and, by inference, how DksA protein expression may be controlled in the cell.